IL-21 shapes germinal center polarization via light zone B cell selection and cyclin D3 upregulation.

Germinal center (GC) dysregulation has been widely reported in the context of autoimmunity. Here, we show that interleukin 21 (IL-21), the archetypal follicular helper T cell (Tfh) cytokine, shapes the scale and polarization of spontaneous chronic autoimmune as well as transient immunization-induced GC. We find that IL-21 receptor deficiency results in smaller GC that are profoundly skewed toward a light zone GC B cell phenotype and that IL-21 plays a key role in selection of light zone GC B cells for entry to the dark zone. Light zone skewing has been previously reported in mice lacking the cell cycle regulator cyclin D3. We demonstrate that IL-21 triggers cyclin D3 upregulation in GC B cells, thereby tuning dark zone inertial cell cycling. Lastly, we identify Foxo1 regulation as a link between IL-21 signaling and GC dark zone formation. These findings reveal new biological roles for IL-21 within GC and have implications for autoimmune settings where IL-21 is overproduced.

Systematic Review of Patient-Reported Outcome Measures in Locally Recurrent Rectal Cancer.

BACKGROUND: The availability of high-quality patient-reported outcome (PRO) data is crucial to guiding shared decision-making in the context of locally recurrent rectal cancer (LRRC), where potential treatment benefits must be balanced against the impact of both the disease and treatment on PROs, such as quality of life. This review aimed to identify the patient-reported outcome measures (PROMs) currently being reported in LRRC and to appraise the methodological quality of studies using these measures. METHODS: PubMed, Embase and CINAHL databases were searched, including studies published up until 14th September 2022. Studies in adults with LRRC reporting PROMS as a primary or secondary outcome measure were included. Data were extracted concerning the methodological quality of the reporting of PROMs using criteria informed by the CONSORT-PRO checklist and the psychometric properties of the PROMs identified using the COSMIN Risk of Bias checklist. RESULTS: Thirty-five studies including 1914 patients with LRRC were identified. None of the studies included in the review met all eleven criteria for the quality of reporting of PROMs. Seventeen PROMs and two clinician-reported outcome measures were identified, none of which have been validated for use in patients with LRRC. CONCLUSIONS: None of the PROMs which are currently being used to report PROs in LRRC have been validated for use in this cohort of patients. Future studies in this disease area should focus on utilising PROMs that have undergone a robust development process including patients with LRRC, to produce data which is high quality, accurate and relevant.

Stratification of PD-1 blockade response in melanoma using pre- and post-treatment immunophenotyping of peripheral blood.

Efficacy of checkpoint inhibitor therapies in cancer varies greatly, with some patients showing complete responses while others do not respond and experience progressive disease. We aimed to identify correlates of response and progression following PD-1-directed therapy by immunophenotyping peripheral blood samples from 20 patients with advanced malignant melanoma before and after treatment with the PD-1 blocking antibody pembrolizumab. Our data reveal that individuals responding to PD-1 blockade were characterised by increased CD8 T cell proliferation following treatment, while progression was associated with an increase in CTLA-4-expressing Treg. Remarkably, unsupervised clustering analysis of pre-treatment T cell subsets revealed differences in individuals that went on to respond to PD-1 blockade compared to individuals that did not. These differences mapped to expression of the proliferation marker Ki67 and the costimulatory receptor CD28 as well as the inhibitory molecules 2B4 and KLRG1. While these results require validation in larger patient cohorts, they suggest that flow cytometric analysis of a relatively small number of T cell markers in peripheral blood could potentially allow stratification of PD-1 blockade treatment response prior to therapy initiation.

Costimulation blockade in combination with IL-2 permits regulatory T cell sparing immunomodulation that inhibits autoimmunity.

Blockade of CD28 costimulation with CTLA-4-Ig/Abatacept is used to dampen effector T cell responses in autoimmune and transplantation settings. However, a significant drawback of this approach is impaired regulatory T cell homeostasis that requires CD28 signaling. Therefore, strategies that restrict the effects of costimulation blockade to effector T cells would be advantageous. Here we probe the relative roles of CD28 and IL-2 in maintaining Treg. We find provision of IL-2 counteracts the regulatory T cell loss induced by costimulation blockade while minimally affecting the conventional T cell compartment. These data suggest that combining costimulation blockade with IL-2 treatment may selectively impair effector T cell responses while maintaining regulatory T cells. Using a mouse model of autoimmune diabetes, we show combined therapy supports regulatory T cell homeostasis and protects from disease. These findings are recapitulated in humanised mice using clinically relevant reagents and provide an exemplar for rational use of a second immunotherapy to offset known limitations of the first.

Follicular helper T cell profiles predict response to costimulation blockade in type 1 diabetes.

Follicular helper T (TFH) cells are implicated in type 1 diabetes (T1D), and their development has been linked to CD28 costimulation. We tested whether TFH cells were decreased by costimulation blockade using the CTLA-4-immunoglobulin (Ig) fusion protein (abatacept) in a mouse model of diabetes and in individuals with new-onset T1D. Unbiased bioinformatics analysis identified that inducible costimulatory molecule (ICOS)+ TFH cells and other ICOS+ populations, including peripheral helper T cells, were highly sensitive to costimulation blockade. We used pretreatment TFH profiles to derive a model that could predict clinical response to abatacept in individuals with T1D. Using two independent approaches, we demonstrated that higher frequencies of ICOS+ TFH cells at baseline were associated with a poor clinical response following abatacept administration. Therefore, TFH analysis may represent a new stratification tool, permitting the identification of individuals most likely to benefit from costimulation blockade.

CTLA-4-mediated transendocytosis of costimulatory molecules primarily targets migratory dendritic cells.

CTLA-4 is a critical negative regulator of the immune system and a major target for immunotherapy. However, precisely how it functions in vivo to maintain immune homeostasis is not clear. As a highly endocytic molecule, CTLA-4 can capture costimulatory ligands from opposing cells by a process of transendocytosis (TE). By restricting costimulatory ligand expression in this manner, CTLA-4 controls the CD28-dependent activation of T cells. Regulatory T cells (Tregs) constitutively express CTLA-4 at high levels and, in its absence, show defects in TE and suppressive function. Activated conventional T cells (Tconv) are also capable of CTLA-4-dependent TE; however, the relative use of this mechanism by Tregs and Tconv in vivo remains unclear. Here, we set out to characterize both the perpetrators and cellular targets of CTLA-4 TE in vivo. We found that Tregs showed constitutive cell surface recruitment of CTLA-4 ex vivo and performed TE rapidly after TCR stimulation. Tregs outperformed activated Tconv at TE in vivo, and expression of ICOS marked Tregs with this capability. Using TCR transgenic Tregs that recognize a protein expressed in the pancreas, we showed that the presentation of tissue-derived self-antigen could trigger Tregs to capture costimulatory ligands in vivo. Last, we identified migratory dendritic cells (DCs) as the major target for Treg-based CTLA-4-dependent regulation in the steady state. These data support a model in which CTLA-4 expressed on Tregs dynamically regulates the phenotype of DCs trafficking to lymph nodes from peripheral tissues in an antigen-dependent manner.

T Cell/B Cell Collaboration and Autoimmunity: An Intimate Relationship.

Co-ordinated interaction between distinct cell types is a hallmark of successful immune function. A striking example of this is the carefully orchestrated cooperation between helper T cells and B cells that occurs during the initiation and fine-tuning of T-cell dependent antibody responses. While these processes have evolved to permit rapid immune defense against infection, it is becoming increasingly clear that such interactions can also underpin the development of autoimmunity. Here we discuss a selection of cellular and molecular pathways that mediate T cell/B cell collaboration and highlight how in vivo models and genome wide association studies link them with autoimmune disease. In particular, we emphasize how CTLA-4-mediated regulation of CD28 signaling controls the engagement of secondary costimulatory pathways such as ICOS and OX40, and profoundly influences the capacity of T cells to provide B cell help. While our molecular understanding of the co-operation between T cells and B cells derives from analysis of secondary lymphoid tissues, emerging evidence suggests that subtly different rules may govern the interaction of T and B cells at ectopic sites during autoimmune inflammation. Accordingly, the phenotype of the T cells providing help at these sites includes notable distinctions, despite sharing core features with T cells imparting help in secondary lymphoid tissues. Finally, we highlight the interdependence of T cell and B cell responses and suggest that a significant beneficial impact of B cell depletion in autoimmune settings may be its detrimental effect on T cells engaged in molecular conversation with B cells.

Foreign bodies in sigmoid colon diverticulosis.

This review addresses the management of sigmoid colon diverticular disease associated with foreign bodies. In addition, two novel cases are presented. One case describes the management of diverticular bleeding secondary to a chicken bone and the other case reports retrieval of a retained EndoRings™ Device. The review identified 40 relevant publications including 50 subjects. Foreign bodies within sigmoid diverticular disease may be associated with inflammation, perforation, abscess and fistula. In current practice, diagnosis is often achieved with CT scan. Patients with colonic perforation or fistula generally require colonic resection. Patients with inflammation may merit conservative management, including colonoscopic foreign body retrieval. Chicken bones, tooth picks, and biliary stents have been reported in patients with inflammation, perforation and fistula, whereas all published patients with fish bone related diverticulosis complications experienced inflammation. Treatment might be best guided by the consequences of the foreign body rather than the nature of the underlying retained object. Diverticular bleeding secondary to a chicken bone was diagnosed at CT angiography and treated with colonoscopic snare retrieval of the bone and clipping of the bleeding diverticulum. The EndoRings™ Device was retrieved with a colonoscopic balloon.

Sorting nexin 5 selectively regulates dorsal-ruffle-mediated macropinocytosis in primary macrophages.

The regulation of macropinocytosis, a specialised endocytosis pathway, is important for immune cell function. However, it is not known whether the biogenesis of macropinosomes involves one or more distinct pathways. We previously identified sorting nexin 5 (SNX5) as a regulator of macropinocytosis in macrophages. Here, we show that bone-marrow-derived macrophages from SNX5-knockout mice had a 60-70% reduction in macropinocytic uptake of dextran or ovalbumin, whereas phagocytosis and retrograde transport from the plasma membrane to the Golgi was unaffected. In contrast, deficiency of SNX5 had no effect on macropinocytosis or antigen presentation by dendritic cells. Activation of macrophages with CSF-1 resulted in a localisation of SNX5 to actin-rich ruffles in a manner dependent on receptor tyrosine kinases. SNX5-deficient macrophages showed a dramatic reduction in ruffling on the dorsal surface following CSF-1 receptor activation, whereas peripheral ruffling and cell migration were unaffected. We demonstrate that SNX5 is acting upstream of actin polymerisation following CSF-1 receptor activation. Overall, our findings reveal the important contribution of dorsal ruffing to receptor-activated macropinocytosis in primary macrophages and show that SNX5 selectively regulates macropinosomes derived from the dorsal ruffles.

Transient Treg-cell depletion in adult mice results in persistent self-reactive CD4(+) T-cell responses.

Depletion of Foxp3(+) CD4(+) regulatory T cells (Treg) in adults results in chronic inflammation and autoimmune disease. However, the impact of transient Treg-cell depletion on self-reactive responses is poorly defined. Here, we studied the effect of transient depletion of Treg cells on CD4(+) T-cell responses to endogenous self-antigens. Short-term ablation of Treg cells in mice resulted in rapid activation of CD4(+) T cells, increased percentage of IFN-γ(+) and Th17 cells in lymphoid organs, and development of autoimmune gastritis. To track self-reactive responses, we analyzed the activation of naïve gastric-specific CD4(+) T cells. There was a dramatic increase in proliferation and acquisition of effector function of gastric-specific T cells in the stomach draining LNs of Treg-cell-depleted mice, compared with untreated mice, either during Treg-cell depletion or after Treg-cell reconstitution. Moreover, the hyperproliferation of gastric-specific T cells in the Treg-cell-ablated mice was predominantly antigen-dependent. Transient depletion of Treg cells resulted in a shift in the ratio of peripheral:thymic Treg cells in the reemerged Treg-cell population, indicating an altered composition of Treg cells. These findings indicate that transient Treg-cell depletion results in ongoing antigen-driven self-reactive T-cell responses and emphasize the continual requirement for an intact Treg-cell population.

Transient systemic inflammation does not alter the induction of tolerance to gastric autoantigens by migratory dendritic cells.

It has been proposed that activation of dendritic cells (DCs) presenting self-antigens during inflammation may lead to activation of autoreactive T cells and the development of autoimmunity. To test this hypothesis, we examined the presentation of the autoantigen recognized in autoimmune gastritis, gastric H(+)/K(+) ATPase, which is naturally expressed in the stomach and is constitutively presented in the stomach-draining lymph nodes. Systemic administration to mice of the TLR9 agonist CpG DNA, agonist anti-CD40 Ab, or TLR4 agonist LPS all failed to abrogate the process of peripheral clonal deletion of H(+)/K(+) ATPase-specific CD4 T cells or promote the development of autoimmune gastritis. We demonstrated that migratory DCs from the stomach-draining lymph nodes are the only DC subset capable of constitutively presenting the endogenous gastric H(+)/K(+) ATPase autoantigen in its normal physiological context. Analysis of costimulatory molecules indicated that, relative to resident DCs, migratory DCs displayed a partially activated phenotype in the steady state. Furthermore, migratory DCs were refractory to stimulation by transient exposure to TLR agonists, as they failed to upregulate costimulatory molecules, secrete significant amounts of inflammatory cytokines, or induce differentiation of effector T cells. Together, these data show that transient systemic inflammation failed to break tolerance to the gastric autoantigen, as migratory DCs presenting the gastric autoantigen remain tolerogenic under such conditions, demonstrating the robust nature of peripheral tolerance.

Helios defines T cells being driven to tolerance in the periphery and thymus.

The expression of the Ikaros transcription factor family member, Helios, has been shown to be associated with T-cell tolerance in both the thymus and the periphery. To better understand the importance of Helios in tolerance pathways, we have examined the expression of Helios in TCR-transgenic T cells specific for the gastric H(+) /K(+) ATPase, the autoantigen target in autoimmune gastritis. Analysis of H(+) /K(+) ATPase-specific T cells in mice with different patterns of H(+) /K(+) ATPase expression revealed that, in addition to the expression of Helios in CD4(+) Foxp3(+) regulatory T (Treg) cells, Helios is expressed by a large proportion of CD4(+) Foxp3(-) T cells in both the thymus and the paragastric lymph node (PgLN), which drains the stomach. In the thymus, Helios was expressed by H(+) /K(+) ATPase-specific thymocytes that were undergoing negative selection. In the periphery, Helios was expressed in H(+) /K(+) ATPase-specific CD4(+) T cells following H(+) /K(+) ATPase presentation and was more highly expressed when T-cell activation occurred in the absence of inflammation. Analysis of purified H(+) /K(+) ATPase-specific CD4(+) Foxp3(-) Helios(+) T cells demonstrated that they were functionally anergic. These results demonstrate that Helios is expressed by thymic and peripheral T cells that are being driven to tolerance in response to a genuine autoantigen.

Functional outcomes after limb-salvage surgery and endoprosthetic reconstruction with an expandable prosthesis: a report of 4 cases.

OBJECTIVE: To determine the functional outcomes of skeletally immature patients after replacement of the femur and tibia performed by using noninvasive expandable endoprostheses. DESIGN: Case series. SETTING: A hospital-based ambulatory care center. PARTICIPANTS: Pediatric patients (N=4) with primary bone tumors of the distal femur and proximal tibia who underwent surgical replacement performed by using the Repiphysis noninvasive expandable endoprosthesis (Wright Medical Technology, Memphis, TN). INTERVENTIONS: Wide resection of bone sarcoma and placement of expandable endoprosthesis. MAIN OUTCOME MEASURES: Musculoskeletal Tumor Society (MSTS) scores were assessed at the beginning of the study and at each follow-up visit. Medical Outcomes Study 36-Item Short-Form Health Survey, Version 2 (SF-36); gait; sit-to-stand transition; and range of motion (ROM) were assessed at an average follow-up of 31.5 months. RESULTS: At an average of 31.5 months postoperative, the SF-36 physical component summary scores lagged behind the national mean, whereas the mental component summary scores were satisfactory. MSTS scores indicated low levels of pain and supports use with high emotional acceptance and walking ability but persisting difficulties with function and gait. Patients also showed altered patterns of sit-to-stand transition including decreased peak vertical force in the operated limb and increased center of mass momentum in a shorter amount of time. Parts of gait functioning were found to be decreased, including gait velocity, stride length, and cadence. Some patients displayed alternate weight-bearing strategies that accompanied increased double-limb support and stance phase during walking. ROM and strength were diminished at both the hip and knee joints in the operated limb and in the nonoperated limb. CONCLUSIONS: Reconstruction with a noninvasive expandable endoprosthesis produces satisfactory functional outcomes in pediatric patients with primary tumors of the bone. Patients in our study displayed some persisting physical difficulties including decreased ROM and strength and altered gait and sit-to-stand patterns, yet they maintained high levels of emotional acceptance and coping.

Postoperative gait velocity and mean knee flexion in stance of ambulatory children with spastic diplegia four years or more after multilevel surgery.

Factors associated with longer-term outcomes of multilevel orthopaedic surgery in ambulatory children with cerebral palsy using a multivariate approach were evaluated using a retrospective pretest-posttest design. The population included 20 ambulatory children with spastic diplegia who had undergone multilevel orthopaedic surgery with a minimum of 4-year interval between a preoperative and a postoperative gait assessment. Multiple regression analysis was used to identify factors associated with postoperative velocity and mean knee flexion in stance. Independent variables included in the regression models were velocity, mean knee flexion in stance, age at preoperative evaluation, Gross Motor Function Classification System level, use of ankle-foot orthoses, leg length, age-adjusted body mass index, number of surgical procedures, and range of motion of hip and knee. Children who demonstrated faster postoperative gait velocity 4 years or more after surgery were younger at the time of initial evaluation, had undergone fewer surgical procedures, had faster preoperative gait velocity, used ankle-foot orthoses postoperatively, and had increased hip extension range of motion postoperatively (R = 0.55). Children who demonstrated greater knee flexion in stance 4 years or more after surgery had undergone more surgical procedures, greater postoperative popliteal angle, and less knee extension range of motion (R = 0.73). This study demonstrates the usefulness of a multivariate approach toward understanding and predicting outcomes. The results of this study will provide clinicians and researchers more information about those factors associated with maintained improvements in the longer term and may be useful for treatment planning.

Utilizing program evaluation as a strategy to promote community change: evaluation of a comprehensive, community-based, family violence initiative.

This paper describes the authors' work in a community that received Federal funding for an integrated system of care to reduce the impact and incidence of exposure to violence for children less than six years of age. The paper includes a review of the conceptual framework that guided the work of the authors and provides a brief overview of the issue of family violence, the impact of this violence on young children, and the Federal response to this issue. In addition, a description of the Initiative and the community in which it was based is provided along with some aspects of the evaluation plan. Finally, the authors discuss how their work with this Initiative depicts an approach to facilitating change within communities.

Development and evaluation of an interactive multimedia clinical skills teaching program designed for the pediatric clerkship.

BACKGROUND AND PURPOSE: We evaluated the physical-examination section of a multimedia program developed to teach infant history and physical-examination skills. METHODS: A total of 71 students participated: one group viewed only the physical-examination section (PX), one the history section (HX), one none of the program (CX). We assessed physical-examination skills by direct observation of medical students performing an abdominal exam and scored using a checklist at baseline, immediately after intervention, and at the end of the pediatric clerkship. We analyzed results using analysis of variance with repeated measures. RESULTS: Baseline scores were PX = 2.5, HX = 2.8. The PX group scored significantly higher immediately postintervention at 6.8 compared to the HX group (3.1). At the end of the clerkship, significant differences between the groups remained. Final group mean scores were PX = 5.5, HX = 4.4, and CX = 2.7. CONCLUSION: The program improved examination skills with attenuation over 6 weeks.